Molecular and phenotypic characteristics of RSV infections in infants during two nirsevimab randomized clinical trials

Nirsevimab is a monoclonal antibody that binds to the respiratory syncytial virus (RSV) fusion protein. During the Phase 2b (NCT02878330) and MELODY (NCT03979313) clinical trials, infants received one dose of nirsevimab or placebo before their first RSV season. In this pre-specified analysis, isolates from RSV infections were subtyped, sequenced and analyzed for nirsevimab binding site substitutions; subsequently, recombinant RSVs were engineered for microneutralization susceptibility testing. Here we show that the frequency of infections caused by subtypes A and B is similar across and within the two trials. In addition, RSV A had one and RSV B had 10 fusion protein substitutions occurring at >5% frequency. Notably, RSV B binding site substitutions were rare, except for the highly prevalent I206M:Q209R, which increases nirsevimab susceptibility; RSV B isolates from two participants had binding site substitutions that reduce nirsevimab susceptibility. Overall, >99% of isolates from the Phase 2b and MELODY trials retained susceptibility to nirsevimab.

Remarks to the Author: RSV infections are a major medical burden. Since 1198 monthly palivizumab administration has been used to protect high risk infants from severe RSV disease. Nirsevimab is a fully human monoclonal antibody with a marked higher neutralization activity as compared to palivizumab. In a recent Phase III study this antibody was shown to reduce RSV related hospitalization and RSV infections that require medical attention of late-preterm and term infants with about 80%. Due to the high potency of this antibody and its extended half-life this could be accomplished by just a single shot. As such Nirsevimab is intended to be used for the general infant population at a reasonable cost. Although the binding site of nirsevimab is highly conserved, general use of a monoclonal antibody might come with the risk of introducing virus variants that escape from this antibody and possibly also form other antibodies. For RSV A breakthrough infections were not associated with RSV variants that carry mutations at the antibody binding site. In the submitted report, Ahani and colleagues investigated the incidence, genotype and phenotype of RSV variants in breakthrough RSV infections among infants that were treated with nirsevimab or with placebo. For RSV B two viral variants were isolated that had mutations in the binding site that markedly reduced the sensitivity to nirsevimab mediated neutralization. Without doubt this investigation is of major medical relevance and highly appreciated. The results are clearly explained and underscore the conclusions of the authors. There are however some important questions that need to be addressed before publication. My major comment is that isolated RSV B variants should be studied in more detail. In the study the authors isolated form 2 patients treated with nirsevimab two RSV B variants that have a strongly increased resistance to nirsevimab due to mutations in the antibody binding region. The first variant concerns a variant that next to the recurrent I206M:Q209R double mutation acquired I64T+K68E mutation, all of which are located in the antibody binding region. Although the I206M:Q209R mutation increases the sensitivity for nirsevimab, the I64T and K68E respectively resulted in a 496 and 283-fold reduction in neutralization sensitivity. A similar level of reduction in neutralization sensitivity was observed for recombinant viruses carrying the N208S mutation. Because of their impact, these three mutations and the I64T+K68E combination require more in depth analysis. Moreover, although rare in the study cohort, these mutations might increase in frequency when nirsevimab is used much more generally for multiple seasons. One important question that needs to be addressed is to what extend do the I64T and K68E mutations either apart or combined impact the viral replication of the recombinant RSV either WT or harboring the I206M:Q209R mutations. Especially the K68E substitution is a remerkable change. Similarly, what is the impact of the N208S mutation? This can be analyzed by comparing the viral replication kinetics of the respective recombinant RSV variants used in the study (recRSV WT vs recRSV_N208S vs recRSV_I64T vs recRSV_K68E and recRSV_I206M:Q209R vs recRSV_I206M:Q209R+I64T+K68E). A potential reduced fitness of the variants that escape nirsevimab would be reassuring. If clear differences in viral replication do occur, it would be highly informative if this is associated by a difference in fusion activity of the F protein variants. This can be tested by expressing the F protein (eg GFP or splitGFP) variants in mammalian cells and monitor syncytia formation via an reporter (eg co-expression of GFP or splitGFP co-expression,..). As the binding region of nirsevimab is the most important site for neutralizing antibodies, the above described mutations of concern (I64T, K68E and N208S) could also impact neutralization by protective antibodies evoked by infection or preF vaccination. Therefore, these mutations should also be tested for their impact on the neutralizing activity by e.g reference human serum containing high levels of neutralizing antibodies or by post challenge or preF-vaccination animal serum. To my knowledge no viral escape analysis using nirsevimab or the parental antibody (D25 or MEDI8897) has been reported yet. If in contrast such an analysis has been reported, did the above described mutations appear in this analysis? If no escape analysis has been reported the authors should perform an in vitro viral escape selection of recRSV in the presence of nirsevimab and as control palivizumab. Virals escape variants for palivizumab can readily be selected in vitro. The authors should describe the design and production of the used recombinant viruses.
Please discuss the RSV variants that were investigated in the context of the findings of Zhu et al (Science transl med 2017) on the impact of naturally occurring RSV variants in the binding site region on the neutralizing activity of MEDI8897. Please, include for clarity as supplementary table a list of all RSV F variants that have been isolated in this study + their respective frequency in the nirsevimab and placebo arms (i.e for both the nir and placebo arm: number of cases in which a specific variant has been isolated / number of participants in this arm) Reviewer #2: Remarks to the Author: For its extended half-life and locking the RSV F protein in the pre-fusion conformation, Nirsevimab has been proved as a powerful monoclonal antibody to prevent RSV infection. In the research work, the authors tried to investigate nirsevimab binding site substitutions and the phenotypic characteristics of these substitutions during the Phase 2b and MELODY clinical trials. Major concern: 1. Through the whole work, there is no result associated with preterm and term infants. Therefore, the title of the manuscript should be concise. 2. In Abstract, there were "242 RSV isolates collected during the Phase 2b and MELODY clinical trials". However, there were only 105 participants analyzed in the investigation. And no cell lines were used for RSV isolation. The results concluded that "Frequency of infections caused by subtypes A and B was similar across and within the two trials". In fact, statistical results should be provided instead of conclusive sentences, a common problem in the manuscript, also shown in "which increased nirsevimab susceptibility". In the research work, only RSV infection participants with NGS results were subtyped, which cannot present the frequency of infections caused by subtypes A and B. 3. In Material and Method, The participant selection criteria should be special for the study instead of those published previously. Why were the Netherlands RSV A and RSV B 2013 reference strains chosen? The part of "RSV microneutralization susceptibility assay" should be the key part of the manuscript, which should be described in detail. 4. The organization of the contents of Results should be strengthened to make them easier to understand. Important results should be reflected, not just shown in the table or figure. The conclusions with "higher" should be based on statistical results. 5. In Discussion, "This study of infants with confirmed RSV, including LRTI and respiratory illnesses requiring hospitalization, demonstrated that nirsevimab effectively neutralized both RSV A and B infections in the Phase 2b and MELODY trials, including RSV infections that did not meet protocol defined endpoints." is not the conclusion of the research work. And the content of Discussion is too short to deep the significance of the research work.
Reviewer #3: Remarks to the Author: The manuscript from Ahani, Tuffy and colleagues investigates the molecular and phenotypic characteristics of RSV isolates from infants in Phase 2b and Phase III nirsevimab clinical trials. One nirsevimab-binding-site substitution was identified in RSV A (K209R, 2.1%), but this substitution did not affect niresevimab susceptibility. Interestingly, two RSV B substitutions (I206M, Q209R) occurred with a frequency greater than 5% in the Phase 2b and MELODY trials, but these substitutions actually increase susceptibility to nirsevimab. Based on molecular modeling, the investigators provide a reasonable basis for the increased susceptibility, which is due to improved electrostatic interactions resulting from the Q209R substitution. There were, however, additional nirsevimab-binding-site substitutions found in RSV B, including L204S/S211N, I64T/K68E, and N208S. The I64T, K68E, and N208S substitutions were all associated with substantially increased nirsevimab resistance (>200-fold IC50). Unfortunately, the molecular basis for the increased resistance was not provided by the authors.
These are important studies for understanding the extent to which the use of nirsevimab influences RSV substitutions and evolution. The manuscript is succinct and well written, and the conclusions are supported by the data. My only major comment is that the authors are encouraged to provide a molecular basis for the resistance afforded by I64T, K68E, and N208S.
Other comments: 1) The structural figures in 2A are fairly small and general readers may have difficulty interpreting the location of the amino acid substitutions. The pink outline of the nirsevimab binding site is also difficult to discern.
2) The amino acid substitution N208S is not listed in the key for Figure 6. Is this an omission? RSV infections are a major medical burden. Since 1198 monthly palivizumab administration has been used to protect high risk infants from severe RSV disease. Nirsevimab is a fully human monoclonal antibody with a marked higher neutralization activity as compared to palivizumab. In a recent Phase III study this antibody was shown to reduce RSV related hospitalization and RSV infections that require medical attention of late-preterm and term infants with about 80%. Due to the high potency of this antibody and its extended half-life this could be accomplished by just a single shot. As such Nirsevimab is intended to be used for the general infant population at a reasonable cost. Although the binding site of nirsevimab is highly conserved, general use of a monoclonal antibody might come with the risk of introducing virus variants that escape from this antibody and possibly also form other antibodies. For RSV A breakthrough infections were not associated with RSV variants that carry mutations at the antibody binding site.
In the submitted report, Ahani and colleagues investigated the incidence, genotype and phenotype of RSV variants in breakthrough RSV infections among infants that were treated with nirsevimab or with placebo. For RSV B two viral variants were Thank you.
isolated that had mutations in the binding site that markedly reduced the sensitivity to nirsevimab mediated neutralization.
Without doubt this investigation is of major medical relevance and highly appreciated. The results are clearly explained and underscore the conclusions of the authors. There are however some important questions that need to be addressed before publication.
My major comment is that isolated RSV B variants should be studied in more detail. In the study the authors isolated form 2 patients treated with nirsevimab two RSV B variants that have a strongly increased resistance to nirsevimab due to mutations in the antibody binding region. The first variant concerns a variant that next to the recurrent I206M:Q209R double mutation acquired I64T+K68E mutation, all of which are located in the antibody binding region. Although the I206M:Q209R mutation increases the sensitivity for nirsevimab, the I64T and K68E respectively resulted in a 496 and 283-fold reduction in neutralization sensitivity. A similar level of reduction in neutralization sensitivity was observed for recombinant viruses carrying the N208S mutation. Because of their impact, these three mutations and the I64T+K68E combination require more in depth analysis We acknowledge the importance of investigating the impact of the observed substitutions on viral replication. However, these explorations are extensive in scale and are beyond the scope of the current manuscript.
We are planning to perform analyses similar to those proposed, but these findings will be captured in a separate manuscript. Notably, fitness of the Moreover, although rare in the study cohort, these mutations might increase in frequency when nirsevimab is used much more generally for multiple seasons. One important question that needs to be addressed is to what extend do the I64T and K68E mutations The effect that widespread use of nirsevimab will have on the emergence of resistant variants remains unknown. However, we anticipate that the selective pressure applied by nirsevimab usage will be limited by both the overall conservation of antigenic site Ø (limiting the pool from which to either apart or combined impact the viral replication of the recombinant RSV either WT or harboring the I206M:Q209R mutations. Especially the K68E substitution is a remarkable change. be selected) and the infant population (infants <1 year have a large burden of disease but are not responsible for the majority of RSV transmission).
Additionally, while antigenic site Ø is the major target for neutralizing antibodies on Pre-F, it is not the only site. However, in vitro studies did not reveal enhanced growth kinetics of the N208S variant versus the parental reference B9320. A substitution at amino acid position 68 was also described; however, it was a K68N substitution rather than the K68E observed in the current study. This variant also displayed similar growth kinetics to the parent virus. These results have now been described in the discussion, pages 14, lines 309-319 as follows: mammalian cells and monitor syncytia formation via an reporter (eg co-expression of GFP or splitGFP co-expression,..). As the binding region of nirsevimab is the most important site for neutralizing antibodies, the above described mutations of concern (I64T, K68E and N208S) could also impact neutralization by protective antibodies evoked by infection or preF vaccination. Therefore, these mutations should also be tested for their impact on the neutralizing activity by e.g reference human serum containing high levels of neutralizing antibodies or by post challenge or preFvaccination animal serum.

Of the isolates identified from two participants in the
To my knowledge no viral escape analysis using nirsevimab or the parental antibody (D25 or MEDI8897) has been reported yet. If in contrast such an analysis has been reported, did the above Thank you for this helpful suggestion to improve our manuscript. As  These data are included in Supplementary Tables 2-6 which are broken out by the corresponding protocol-defined endpoints.

Reviewer #2
For its extended half-life and locking the RSV F protein in the prefusion conformation, Nirsevimab has been proved as a powerful monoclonal antibody to prevent RSV infection. In the research work, the authors tried to investigate nirsevimab binding site substitutions and the phenotypic characteristics of these substitutions during the Phase 2b and MELODY clinical trials.
Major concern: 1. Through the whole work, there is no result associated with preterm and term infants. Therefore, the title of the manuscript The part of "RSV microneutralization susceptibility assay" should be the key part of the manuscript, which should be described in detail.
Additional details on the microneutralization susceptibility assay previously included in the supplement have been moved to page 7-8, lines 147-160, as follows: Microneutralization assays were conducted by Viroclinics Biosciences BV (Rotterdam, Netherlands)  1. High-level overview of number of cases of RSV, by study and subtype 2. Genotypic analyses of the RSV infections described in Figure 1 3. Analysis of the nirsevimab binding site from the genotypic analyses in Figure 2 4. Phenotypic analysis of all individual substitutions identified in the study, including those seen in Figures 2/3 5. Deep-dive into the two infants who had resistant substitutions identified in Figure 4 6. Deep-dive into the highly prevalent nirsevimab binding site substitution that had higher potency than the reference amino acid 7. Molecular modeling of the binding site substitution that is characterized in Figure 6 that shows the mechanism for the increase in potency This organization is logical to the authors. We have added further description to the results section; however, as per Nature editorial style, we have left interpretation of the data to the Discussion. 5. In Discussion, "This study of infants with confirmed RSV, including LRTI and respiratory illnesses requiring hospitalization, demonstrated that nirsevimab effectively neutralized both RSV A and B infections in the Phase 2b and MELODY trials, including RSV infections that did not meet protocol defined endpoints." is not the conclusion of the research work. And the content of Discussion is too short to deep the significance of the research work.
Thank you for the opportunity to clarify the key messages of our manuscript and further expand the discussion. A tracked changes copy of the manuscript and its updated discussion are included with this response.

Reviewer #3
The manuscript from Ahani, Tuffy and colleagues investigates the molecular and phenotypic characteristics of RSV isolates from infants in Phase 2b and Phase III nirsevimab clinical trials. One nirsevimab-binding-site substitution was identified in RSV A (K209R, 2.1%), but this substitution did not affect nirsevimab susceptibility. Interestingly, two RSV B substitutions (I206M, Q209R) occurred with a frequency greater than 5% in the Phase 2b and MELODY trials, but these substitutions actually increase susceptibility to nirsevimab. Based on molecular modeling, the investigators provide a reasonable basis for the increased susceptibility, which is due to improved electrostatic interactions resulting from the Q209R substitution. There were, however, additional nirsevimab-binding-site substitutions found in RSV B, including L204S/S211N, I64T/K68E, and N208S. The I64T, K68E, and N208S substitutions were all associated with substantially Thank you for the thoughtful review and the opportunity to improve the manuscript. In regards, to the molecular basis of the I64T:K68E and N208S substitutions, we have now performed molecular modeling of these substitutions in RSV F and the analysis is included on page 13, lines 281-288 as follows: A subsequent investigation into the RSV B I64T: K68E and N208S substitutions that were associated with nirsevimab resistance was also performed. I64, K68, and N208 have all been reported to interact with nirsevimab, and the resulting K68E and N208S in silico substitutions led to the loss and reduction of polar contacts, respectively (Supplementary Figure 5). I64 is near the heavy chain CDR3 of nirsevimab and forms a close contact with V99 (<4 Å). However, the I64T in silico substitution did not impact the distance between these residues (Supplementary Figure 5). Instead, the decrease in nirsevimab potency due to this increased nirsevimab resistance (>200-fold IC50). Unfortunately, the molecular basis for the increased resistance was not provided by the authors.
These are important studies for understanding the extent to which the use of nirsevimab influences RSV substitutions and evolution.
The manuscript is succinct and well written, and the conclusions are supported by the data. My only major comment is that the authors are encouraged to provide a molecular basis for the resistance afforded by I64T, K68E, and N208S. substitution may stem from introducing a polar side chain into the site Ønirsevimab interface, thus disrupting a patch of hydrophobic interactions.
Other comments: 1) The structural figures in 2A are fairly small and general readers may have difficulty interpreting the location of the amino acid substitutions. The pink outline of the nirsevimab binding site is also difficult to discern.
The figure has been revised to make the greys paler so that the colors are more prominent/clear. Regarding the size of the figures, this will depend on the final layout, which is beyond the control of the authors.
2) The amino acid substitution N208S is not listed in the key for